Compositions and methods for treating elastic fiber breakdown

ABSTRACT

The present disclosure relates to pharmaceutical compositions capable of regulating elastic fiber. The disclosure further relates to methods of treating elastic fiber-related disorders, such as COPD, by inhalation of low-molecular weight hyaluronic acid.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/656,654, filed Apr. 12, 2018, the content ofwhich is hereby incorporated herein by reference in its entirety.

BACKGROUND

The protease-antiprotease concept of lung interstitial injury hasencouraged the use of elastase inhibitors as potential treatments forchronic obstructive pulmonary disease (COPD). However, aside from α-1antiprotease (AAP) replacement therapy for AAP-deficient patients, thisapproach has met with little success, and clinical testing of variousinhibitors has failed to produce a successful drug for COPD.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the discovery thatinhalation of hyaluronan (HA), a long-chain polysaccharidepreferentially binds to lung elastic fibers, prevents elastolysis, andlimits airspace enlargement in experimental models of pulmonaryemphysema. Accordingly, in certain embodiments, the disclosure providesmethods of treating elastic fiber related disorder by administeringdoses of low-molecular weight hyaluronic acid inhalation solution.

In one aspect, a pharmaceutical composition comprising hyaluronic acid,or a pharmaceutically acceptable salt thereof, wherein the hyaluronicacid has a weight average molecular weight (M_(w)) of about 50 to about1000 kDa; and a pharmaceutically acceptable excipient, is provided. Inanother aspect, an inhaler comprising the pharmaceutical compositiondescribed herein, is provided. In still another aspect, a method fortreating an elastic fiber-related disorder in a subject, comprisingadministering to the subject hyaluronic acid, or a pharmaceuticallyacceptable salt thereof, wherein the hyaluronic acid has a weightaverage molecular weight (M_(w)) of about 50 to about 1000 kDa, isprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a progressive reduction in plasma DID levels followingtreatment with HA (r=−0.98; p=0.02). Data are expressed as mean±SEM.Abbreviations: DID, desmosine and isodesmosine; SEM, standard error ofthe mean.

FIG. 2 shows a statistically significant negative correlation betweensputum DID levels and time following treatment with HA (r=−0.97;p=0.03). Data are expressed as mean SEM. Abbreviations: DID, desmosineand isodesmosine; SEM, standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery thatupon inhalation, hyaluronan (HA), a long-chain polysaccharide,preferentially binds to lung elastic fibers, prevents elastolysis, andlimits airspace enlargement in experimental models of pulmonaryemphysema. Desmosine and isodesmosine (DID) concentrations in plasma,sputum, and urine are measured as markers of elastin degradationsystemically in the lung and also markers of inflammation andfibrinogen.

The results of the study in Example 1 indicate that aerosoladministration of a 0.01% solution of low-molecular weight HA was welltolerated and did not involve adverse events requiring cessation oftreatment. Moreover, there were significant reductions in the levels ofDID in plasma and sputum, suggesting that nebulized HA is protectingelastin fibers from degradation and may therefore limit the developmentof pulmonary emphysema. In comparison to a recent trial involvinglong-term administration of AAP to chronic obstructive pulmonary disease(COPD) patients with AAP deficiency, the reduction in plasma levels ofDID in the current study was greater and occurred more rapidly. Thecontinued drop in both plasma and sputum DID levels during the weekfollowing cessation of treatment reflects the binding of HA to elasticfibers, which persists well beyond its half-life in the lung.

It is theorized that aerosolized fluorescein-labeled HA (150 kDamolecular weight) is able to enter the alveoli and preferentially bindsto alveolar wall elastic fibers, presumably preventing interaction withinjurious agents such as elastases and oxidants.

Aerosolized preparations of HA compounds have sometimes been used astherapeutic aerosols for certain upper respiratory disorders, but havenot been tested for efficacy in other diseases or disorders, such asCOPD. HA has also been used in patients with cystic fibrosis as anadditive to hypertonic saline therapy. The HA used in these therapieshas been prepared in high molecular weight forms (300-1,000 kDa). It istheorized that lower-molecular weight forms of HA, such as 150 kDa HA,have a lower viscosity and can achieve lower aerosol particle diameters,thereby facilitating their access to elastic fibers in the distal lung,whose destruction is a central feature of pulmonary emphysema.

While there are a number of animal studies indicating pro-inflammatoryevents associated with low-molecular-weight HA, the findings aregenerally associated with preexisting acute lung injury, and so cannotbe considered as representative of the chronic, subacute inflammatoryprocess associated with COPD. Moreover, the clinical trial describedherein showed no evidence of HA-induced inflammation, and long-termtreatment of smoke-exposed animals with aerosolized HA did not showincreased numbers of leukocytes, in either bronchoalveolar lavage fluidor histological sections of the lung.

Since elastic fiber breakdown may be a final common pathway in COPD, HAmight be effective against a variety of injurious agents involved in thepathogenesis of this disease. In contrast to other proposed treatments,such as specific elastase inhibitors, HA might provide broaderprotection of the lung with fewer potential side effects. Furthermore,the generally slow progression of alveolar wall damage in COPD suggeststhat even small decreases in elastic fiber injury could significantlyreduce the risk of respiratory failure.

Accordingly, in certain embodiments, the disclosure provides methods oftreating elastic fiber related disorders by administering doses oflow-molecular weight hyaluronic acid, for example as an inhalationsolution. Elastic fibers (or yellow fibers) are bundles of proteins(elastin) found in extracellular matrix of connective tissue andproduced by fibroblasts and smooth muscle cells in arteries. Elasticfibers include elastin, elaunin and oxytalan. Elastic tissue isclassified as “connective tissue proper”. The elastic fiber is formedfrom the elastic microfibril (consisting of numerous proteins such asmicrofibrillar-associated glycoproteins, fibrillin, fibullin, and theelastin receptor) and amorphous elastin. Elastic fibers are found in theskin, lungs, arteries, veins, connective tissue proper, elasticcartilage, periodontal ligament, fetal tissue and other structures.

Non-limiting examples of elastic fiber related disorder are cutis laxa,Williams syndrome, alpha-1 antitrypsin deficiency (elastin isexcessively degraded by elastase), emphysema, chronic obstructivepulmonary disease (COPD), liver disease, Buschke-Ollendorff syndrome,Menkes disease, pseudoxanthoma elasticum, Marfan's syndrome Hurlerdisease, hypertension and congenital heart defects.

In some aspects, a pharmaceutical composition comprising hyaluronicacid, or a pharmaceutically acceptable salt thereof, wherein thehyaluronic acid has a weight average molecular weight (M_(w)) of about50 to about 1000 kDa; and a pharmaceutically acceptable excipient, isprovided.

Numerous embodiments are further provided that can be applied to anyaspect of the present disclosure. For example, in some embodiments, thehyaluronic acid has a weight average molecular weight (M_(w)) of about50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa,about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about240 kDa, about 250 kDa, about 260 kDa, about 270 kDa, about 280 kDa,about 290 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600kDa, about 700 kDa, about 800 kDa, about 900 kDa, and/or about 1000 kDa.In some embodiments, the hyaluronic acid has a weight average molecularweight (M_(w)) of 50-900 kDa, 50-800 kDa, 50-700 kDa, 50-600 kDa, 50-500kDa, 50-400 kDa, 50-300 kDa, 50-290 KDa, 50-280 KDa, 50-270 KDa, 50-260KDa, 50-250 KDa, 50-240 KDa, 50-230 KDa, 50-220 KDa, 50-210 KDa, 50-200KDa, 50-190 KDa, 50-180 KDa, 50-170 KDa, 50-160 KDa, 50-150 KDa, 50-140KDa, 50-130 KDa, 50-120 KDa, 50-110 KDa, or 50-100 KDa. In someembodiments, the hyaluronic acid has a weight average molecular weight(M_(w)) of about 150 kDa.

In some embodiments, the pharmaceutical composition described herein isin a form suitable for inhalation. In some embodiments, thepharmaceutical composition described herein is in a form suitable fornasal, intrapulmomary, intratracheal, intrabronchial, or intra-alveolaradministration. In some embodiments, the pharmaceutical compositiondescribed herein is in a form suitable for intra-alveolaradministration. In some embodiments, the pharmaceutical compositiondescribed herein is in a form suitable for administration through a drypowder inhaler. In some embodiments, the pharmaceutical compositiondescribed herein is in a form suitable for administration through aliquid spray device. In some embodiments, the liquid spray device is anaerosol device. In some embodiments, the aerosol device is a nebulizeror electrohydrodynamic aerosol device.

In some aspects, an inhaler comprising the pharmaceutical compositiondescribed herein, is provided.

Numerous embodiments are further provided that can be applied to anyaspect of the present disclosure. For example, in some embodiments, theinhaler described herein is configured to deliver droplets with a sizeof 0.1-5 μm, 0.1-4 μm, 0.1-3 μm, 0.1-2 μm, 0.1-1 μm, 1-5 μm, 1-4, μm,1-3 μm, 1-2 μm, 2-5 μm, 2-4 μm, 2-3 μm, 3-5 μm, 3-4 μm, or 4-5 μm. Insome embodiments, the inhaler described herein is configured to deliverdroplets with a size of about 0.1 μm, about 0.2 μm, about 0.3 μm, about0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about0.9 μm, about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm,about 3.5 μm, about 41 μm, about 4.5 μm, or about 5 μm. In someembodiments, the inhaler described herein is configured to deliverdroplets with a size of 2 μm to 3 μm.

In some aspects, a method is provided for treating an elasticfiber-related disorder in a subject, comprising administering to thesubject hyaluronic acid, or a pharmaceutically acceptable salt thereof,wherein the hyaluronic acid has a weight average molecular weight(M_(w)) of 50 to 1000 kDa.

Numerous embodiments are further provided that can be applied to anyaspect of the present disclosure. For example, in some embodiments, thehyaluronic acid has a weight average molecular weight (M_(w)) of about50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa,about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about240 kDa, about 250 kDa, about 260 kDa, about 270 kDa, about 280 kDa,about 290 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600kDa, about 700 kDa, about 800 kDa, about 900 kDa, and/or about 1000 kDa.In some embodiments, the hyaluronic acid has a weight average molecularweight (M_(w)) of 50-900 kDa, 50-800 kDa, 50-700 kDa, 50-600 kDa, 50-500kDa, 50-400 kDa, 50-300 kDa, 50-290 KDa, 50-280 KDa, 50-270 KDa, 50-260KDa, 50-250 KDa, 50-240 KDa, 50-230 KDa, 50-220 KDa, 50-210 KDa, 50-200KDa, 50-190 KDa, 50-180 KDa, 50-170 KDa, 50-160 KDa, 50-150 KDa, 50-140KDa, 50-130 KDa, 50-120 KDa, 50-110 KDa, or 50-100 KDa. In someembodiments, the hyaluronic acid has a weight average molecular weight(M_(w)) of about 150 kDa. In some embodiments, the method describedherein comprising administering the hyaluronic acid via inhalation. Insome embodiments, the method described herein comprising administeringthe hyaluronic acid to the paranasal sinuses, the pulmonary system, thetrachea, the bronchia, or the alveoli. In some embodiments, the elasticfiber-related disorder is cutis laxa, Williams syndrome, alpha-1antitrypsin deficiency, emphysema, Chronic Obstructive Pulmonary Disease(COPD), liver disease, Buschke-Ollendorff syndrome, Menkes disease,pseudoxanthoma elasticum, Marfan's syndrome, Hurler disease,hypertension or a congenital heart defect. In some embodiments, theelastic fiber-related disorder is COPD.

Pharmaceutical Compositions

The compositions and methods of the present disclosure may be utilizedto treat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a compound of the disclosure (e.g., hyaluronicacid of an appropriate molecular weight as disclosed herein) and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as water or physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oil,or injectable organic esters. In preferred embodiments, when suchpharmaceutical compositions are for human administration, particularlyfor invasive routes of administration, the aqueous solution ispyrogen-free, or substantially pyrogen-free. The excipients can bechosen, for example, to effect delayed release of an agent or toselectively target one or more cells, tissues or organs. Thepharmaceutical composition can be in dosage unit form such as tablet,capsule (including sprinkle capsule and gelatin capsule), granule,lyophile for reconstitution, powder, aerosol, solution, syrup,suppository, injection or the like. The composition can also be presentin a transdermal delivery system, e.g., a skin patch. The compositioncan also be present in a solution suitable for inhalationadministration, such as a sprayed into the nose and absorbed through thenasal membranes (nasally) and/or Breathed into the lungs, through themouth (by inhalation) or mouth and nose (by nebulization).

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the disclosure. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a self emulsifying drug deliverysystem or a self microemulsifying drug delivery system. Thepharmaceutical composition (preparation) also can be a liposome or otherpolymer matrix, which can have incorporated therein, for example, acompound of the disclosure. Liposomes, for example, which comprisephospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); subcutaneously; transdermally (for example as a patchapplied to the skin); and topically (for example, as a cream, ointmentor spray applied to the skin). The compound may also be formulated forinhalation. In certain embodiments, a compound may be simply dissolvedor suspended in sterile water. Details of appropriate routes ofadministration and compositions suitable for same can be found in, forexample, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231,5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 0.001 percent toabout ninety-nine percent of active ingredient, preferably from about0.01 percent to about 0.05 percent, most preferably from about 0.01percent to about 0.03 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe disclosure, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the disclosure withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intraocular (such as intravitreal),intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal andintrasternal injection and infusion. Pharmaceutical compositionssuitable for parenteral administration comprise one or more activecompounds in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods provided in the present disclosure, activecompounds can be given per se or as a pharmaceutical compositioncontaining, for example, 0.01 to 99.5% (more preferably, 0.5 to 90%) ofactive ingredient in combination with a pharmaceutically acceptablecarrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices.

Various slow release polymeric devices have been developed and tested invivo in recent years for the controlled delivery of drugs, includingproteinaceous biopharmaceuticals. A variety of biocompatible polymers(including hydrogels), including both biodegradable and non-degradablepolymers, can be used to form an implant for the sustained release of acompound at a particular target site.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the disclosure. A larger total dosecan be delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the disclosure will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentdisclosure, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans; and other mammals such as equines,cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

In certain embodiments, compounds of the disclosure may be used alone orconjointly administered with another type of therapeutic agent.

The present disclosure includes the use of pharmaceutically acceptablesalts of compounds of the disclosure in the compositions and methods ofthe present disclosure. In certain embodiments, contemplated salts ofthe disclosure include, but are not limited to, alkyl, dialkyl, trialkylor tetra-alkyl ammonium salts. In certain embodiments, contemplatedsalts of the disclosure include, but are not limited to, L-arginine,benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol,diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine,potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,tromethamine, and zinc salts. In certain embodiments, contemplated saltsof the disclosure include, but are not limited to, Na, Ca, K, Mg, Zn orother metal salts. In certain embodiments, contemplated salts of thedisclosure include, but are not limited to, 1-hydroxy-2-naphthoic acid,2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaricacid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid,adipic acid, I-ascorbic acid, 1-aspartic acid, benzenesulfonic acid,benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capricacid (decanoic acid), caproic acid (hexanoic acid), caprylic acid(octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, formic acid, fumaric acid, galactaric acid, gentisic acid,d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid,glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid,hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid,lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid,mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid,oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionicacid, 1-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid,succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acidsalts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In certain embodiments, the present disclosure is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the disclosure, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof a disease or disorder contemplated in the disclosure.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for any suitable mode of administration, known tothe art. The pharmaceutical preparations may be sterilized and ifdesired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., analgesic agents.

Routes of administration of any of the compositions of the disclosureinclude nasal, inhalational, intratracheal, intrapulmonary,intrabronchial, and inhalation.

Suitable compositions and dosage forms include, for example,dispersions, suspensions, solutions, syrups, granules, beads, powders,pellets, liquid sprays for nasal or oral administration, dry powder oraerosolized formulations for inhalation, and the like. It should beunderstood that the formulations and compositions that would be usefulin the present disclosure are not limited to the particular formulationsand compositions that are described herein.

Powdered and granular formulations of a pharmaceutical preparation ofthe disclosure may be prepared using known methods. Such formulationsmay be administered directly to a subject, used, for example, to form amaterial that is suitable to administration to a subject. Each of theseformulations may further comprise one or more of dispersing or wettingagent, a suspending agent, and a preservative. Additional excipients,such as fillers and sweetening, flavoring, or coloring agents, may alsobe included in these formulations.

Pharmaceutical compositions of the present disclosure may be prepared,packaged, or sold in a formulation suitable for pulmonary administrationvia the buccal cavity. Such compositions may comprise dry particles thatcomprise the active ingredient and have a diameter in the range fromabout 0.5 to about 7 nanometers, and in certain embodiments from about 1to about 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. In certainembodiments, such powders comprise particles wherein at least 98% of theparticles by weight have a diameter greater than 0.5 nanometers and atleast 95% of the particles by number have a diameter less than 7nanometers. In certain embodiments, at least 95% of the particles byweight have a diameter greater than 1 nanometer and at least 90% of theparticles by number have a diameter less than 6 nanometers. Dry powdercompositions may include a solid fine powder diluent such as sugar andare conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent (incertain embodiments having a particle size of the same order asparticles comprising the active ingredient).

Pharmaceutical compositions of the present disclosure formulated forpulmonary delivery may also provide the active ingredient in the form ofdroplets of a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration in certain embodiments have anaverage diameter in the range from about 0.1 to about 200 nanometers. Insome embodiments, the droplets provided by this route of administrationinclude a suitable amount of droplets (e.g., an amount of dropletssufficient to deliver a therapeutically effective dose to a targettissue as described elsewhere herein) with a diameter of 0.1-5 μm, 0.1-4μm, 0.1-3 μm, 0.1-2 μm, 0.1-1 μm, 1-5 μm, 1-4, μm, 1-3 μm, 1-2 μm, 2-5μm, 2-4 μm, 2-3 μm, 3-5 μm, 3-4 μm, or 4-5 μm. In certain embodiments,this route of administration provides a suitable amount of droplets witha diameter of about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm,about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm,about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about3.5 μm, about 41 μm, about 4.5 μm, or about 5 μm. In certainembodiments, this route of administration provides a suitable amount ofdroplets with a diameter from about 2 μm to about 3 μm.

The pharmaceutical compositions of the present disclosure may bedelivered using an inhalator such as those recited in U.S. Pat. No.8,333,192 B2, which is incorporated herein by reference in its entirety.

The present disclosure provides methods for delivering medicationsdeeper into the lungs and to the medications' pulmonary targets, whichinclude bronchioles and alveoli. It is contemplated that a medicationincludes any particle, molecule or composition administered to asubject, human or animal, to achieve any desired result. For example, anaerosolized medication may be administered into a patient's respiratorytract, wherein the patient may be any animal or human subject. In someembodiments, an aerosolized surfactant is administered into thepatient's respiratory tract that facilitates delivery of the aerosolizedmedication of the first step to the medication's pulmonary target. It iscontemplated that, throughout this specification, the term “particle” or“particles” includes mixtures containing the medication or surfactant,droplets in which the medications or surfactants have been dissolved,solid medication or surfactant particles, and any other compositioncontemplated by one skilled in the art to contain the medication orsurfactant being administered to the patient. The particles or dropletsdeposit on the luminal surface. The location for deposit of theparticles depends on the inhaler characteristics and patient technique.The patient may then inhale a dose of the aerosolized surfactant from asecond inhaler. The same effect can be obtained through the use of atandem inhaler apparatus, or any other application that allows thesmaller particles containing the medications to be delivered.

The compositions may also be administered directly to the lung byinhalation. Any suitable inhaler may be used, for example thosedisclosed in Tong et al., U.S. Pat. No. 6,251,941; Clark et al., U.S.Pat. No. 6,655,379, which are herein incorporated by reference. Foradministration by inhalation, the compositions may be convenientlydelivered to the lung by a number of different devices. For example, aMetered Dose Inhaler (“MDI”) which utilizes canisters that contain asuitable low boiling propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas may be used to deliver the composition directly tothe lung. MDI devices are available from a number of suppliers such as3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories,Glaxo-Wellcome, Schering Plough and Vectura.

Alternatively, a Dry Powder Inhaler (DPI) device may be used toadminister the composition to the lung (For suitable inhalers, see,e.g., Raleigh et al., Proc. Amer. Assoc. Cancer Research Annual Meeting,1999, 40, 397, which is herein incorporated by reference). DPI devicestypically use a mechanism such as a burst of gas to create a cloud ofdry powder inside a container, which may then be inhaled by the patient.DPI devices are also well known in the art and may be purchased from anumber of vendors which include, for example, Fisons, Glaxo-Wellcome,Inhale Therapeutic Systems, ML Laboratories, Qdose and Vectura. Apopular variation is the multiple dose DPI (“MDDPI”) system, whichallows for the delivery of more than one therapeutic dose. MDDPI devicesare available from companies such as AstraZeneca, GlaxoWellcome, IVAX,Schering Plough, SkyePharma and Vectura. For example, capsules andcartridges of gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch for these systems.

Another type of device that may be used to deliver the composition tothe lung is a liquid spray device supplied, for example, by AradigmCorporation. Liquid spray systems use extremely small nozzle holes toaerosolize liquid drug formulations that may then be directly inhaledinto the lung.

In some preferred embodiments, a nebulizer device is used to deliver thecomposition to the lung. Nebulizers create aerosols from liquid drugformulations by using, for example, ultrasonic energy to form fineparticles that may be readily inhaled (See e.g., Verschoyle et al.,British J. Cancer, 1999, 80, Suppl 2, 96, which is herein incorporatedby reference). Examples of nebulizers include devices supplied bySheffield/Systemic Pulmonary Delivery Ltd. (See, Armer et al., U.S. Pat.No. 5,954,047; van der Linden et al., U.S. Pat. No. 5,950,619; van derLinden et al., U.S. Pat. No. 5,970,974, which are herein incorporated byreference), Aventis and Batelle Pulmonary Therapeutics.

In some preferred embodiments, an electrohydrodynamic (“EHD”) aerosoldevice is used to deliver the composition to the lung. EHD aerosoldevices use electrical energy to aerosolize liquid drug solutions orsuspensions (see e.g., Noakes et al., U.S. Pat. No. 4,765,539; Coffee,U.S. Pat. No. 4,962,885; Coffee, U.S. Pat. No. 6,105,877; Coffee, U.S.Pat. No. 6,105,571; Coffee, PCT Application, WO 95/26234, Coffee, PCTApplication, WO 95/26235, Coffee, PCT Application, WO 95/32807, whichare herein incorporated by reference). The electrochemical properties ofthe composition formulation may be important parameters to optimize whendelivering this drug to the lung with an EHD aerosol device and suchoptimization is routinely performed by one of skill in the art. EHDaerosol devices may more efficiently delivery drugs to the lung thanexisting pulmonary delivery technologies. Other suitable methods ofintra-pulmonary delivery of the composition are within the scope of thedisclosure.

Liquid drug formulations suitable for use with nebulizers and liquidspray devices and EHD aerosol devices will typically include thecomposition with a pharmaceutically acceptable carrier. Preferably, thepharmaceutically acceptable carrier is a liquid such as alcohol, water,polyethylene glycol or a perfluorocarbon. Optionally, another materialmay be added to alter the aerosol properties of the solution orsuspension of the composition. Preferably, this material is liquid suchas an alcohol, glycol, polyglycol or a fatty acid. Other methods offormulating liquid drug solutions or suspension suitable for use inaerosol devices are known to those of skill in the art (See, e.g.,Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No. 5,556,611,which are herein incorporated by reference)

In certain embodiments, the composition of the disclosure comprises astable dry powder blend containing levothyroxine sodium hydrate; lactoseparticles, comprising lactose H₂O, gelatin and starch maize; sodiumstarch glycolate; magnesium stearate; and talc silicified, comprisingtalc purified and colloidal silicon dioxide. In other embodiments, thedry powder comprises levothyroxine sodium is in an amount 4 to 0.02 mgper 100 mg of the dry powder. In yet other embodiments, the dry powdercomprises lactose in an amount higher than 90 mg per 100 mg of the drypowder preparation. In yet other embodiments, the dry powder compriseslactose particles consisting of lactose H₂O, gelatin and starch maize,wherein the ratio by weight-mg of: “lactose H₂O”:“gelatin”:“starchmaize” is 55-75:0.20-0.80:20-40. In yet other embodiments, the drypowder comprises sodium starch glycolate in an amount of 4-8 mg per 100mg of dry powder. In yet other embodiments, the dry powder comprisesmagnesium stearate in an amount of 0.5-2 mg per 100 mg of dry powder. Inyet other embodiments, the dry powder comprises talc silicified, in anamount of 2 mg per 100 mg of dry powder, wherein the talc silicifiedcomprises talc purified and colloidal silicon dioxide in an amount of0.667 mg of talc purified and 1.333 mg of colloidal silicon dioxide for2 mg of talc silicified. In yet other embodiments, the blend furthercomprises a lake. In yet other embodiments, the dry powder comprisessodium starch glycolate in an amount of 5-6 mg per 100 mg of dry powder.In yet other embodiments, the dry powder comprises magnesium stearate inan amount of 1 mg per 100 mg of dry powder.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the disclosure.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

As used herein, the singular forms “a”, “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Further, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”The transitional terms “consist” and any grammatical variations thereof,are intended to be limited to the elements stated in the claims andexclude any elements not stated in the claims. The phrases “consistingessentially of” and any grammatical variant thereof indicate that theclaim encompasses embodiments containing the specified elements andincludes additional elements that do not materially affect the basic andnovel characteristic(s) of the claim.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system, and theconventional variability accepted in the art for the concernedparameter.

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, cell and tissue culture,molecular biology, cell and cancer biology, neurobiology,neurochemistry, virology, immunology, microbiology, pharmacology,genetics and protein and nucleic acid chemistry, described herein, arethose well known and commonly used in the art.

The methods and techniques of the present disclosure are generallyperformed, unless otherwise indicated, according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout thisspecification. See, e.g. “Principles of Neural Science”, McGraw-HillMedical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”,Oxford University Press, Inc. (1995); Lodish et al., “Molecular CellBiology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths etal., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co.,N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”,Sinauer Associates, Inc., Sunderland, Mass. (2000).

Chemistry terms used herein, unless otherwise defined herein, are usedaccording to conventional usage in the art, as exemplified by “TheMcGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill,San Francisco, C.A. (1985).

All of the above, and any other publications, patents and publishedpatent applications referred to in this application are specificallyincorporated by reference herein. In case of conflict, the presentspecification, including its specific definitions, will control.

The term “agent” is used herein to denote a chemical compound (such asan organic or inorganic compound, a mixture of chemical compounds), abiological macromolecule (such as a nucleic acid, an antibody, includingparts thereof as well as humanized, chimeric and human antibodies andmonoclonal antibodies, a protein or portion thereof, e.g., a peptide, alipid, a carbohydrate), or an extract made from biological materialssuch as bacteria, plants, fungi, or animal (particularly mammalian)cells or tissues. Agents include, for example, agents whose structure isknown, and those whose structure is not known.

A “patient,” “subject,” or “individual” are used interchangeably andrefer to either a human or a non-human animal. These terms includemammals, such as humans, primates, livestock animals (including bovines,porcines, etc.), companion animals (e.g., canines, felines, etc.) androdents (e.g., mice and rats).

“Treating” a condition or patient refers to taking steps to obtainbeneficial or desired results, including clinical results. As usedherein, and as well understood in the art, “treatment” is an approachfor obtaining beneficial or desired results, including clinical results.Beneficial or desired clinical results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions,diminishment of extent of disease, stabilized (i.e. not worsening) stateof disease, preventing spread of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount.

“Administering” or “administration of” a substance, a compound or anagent to a subject can be carried out using one of a variety of methodsknown to those skilled in the art. For example, a compound or an agentcan be administered, intravenously, arterially, intradermally,intramuscularly, intraperitoneally, subcutaneously, ocularly,sublingually, orally (by ingestion), intranasally (by inhalation),intraspinally, intracerebrally, and transdermally (by absorption, e.g.,through a skin duct). A compound or agent can also appropriately beintroduced by rechargeable or biodegradable polymeric devices or otherdevices, e.g., patches and pumps, or formulations, which provide for theextended, slow or controlled release of the compound or agent.Administering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods.

As used herein, the phrase “conjoint administration” refers to any formof administration of two or more different therapeutic agents such thatthe second agent is administered while the previously administeredtherapeutic agent is still effective in the body (e.g., the two agentsare simultaneously effective in the patient, which may includesynergistic effects of the two agents). For example, the differenttherapeutic compounds can be administered either in the same formulationor in separate formulations, either concomitantly or sequentially. Thus,an individual who receives such treatment can benefit from a combinedeffect of different therapeutic agents.

A “therapeutically effective amount” or a “therapeutically effectivedose” of a drug or agent is an amount of a drug or an agent that, whenadministered to a subject will have the intended therapeutic effect. Thefull therapeutic effect does not necessarily occur by administration ofone dose, and may occur only after administration of a series of doses.Thus, a therapeutically effective amount may be administered in one ormore administrations. The precise effective amount needed for a subjectwill depend upon, for example, the subject's size, health and age, andthe nature and extent of the condition being treated, such as cancer orMDS. The skilled worker can readily determine the effective amount for agiven situation by routine experimentation.

The term “modulate” as used herein includes the inhibition orsuppression of a function or activity (such as cell proliferation) aswell as the enhancement of a function or activity.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, excipients, adjuvants,polymers and other materials and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” or “salt” is used herein to refer toan acid addition salt or a basic addition salt which is suitable for orcompatible with the treatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used hereinmeans any non-toxic organic or inorganic salt of any base compoundsrepresented by Formula I. Illustrative inorganic acids which formsuitable salts include hydrochloric, hydrobromic, sulfuric andphosphoric acids, as well as metal salts such as sodium monohydrogenorthophosphate and potassium hydrogen sulfate. Illustrative organicacids that form suitable salts include mono-, di-, and tricarboxylicacids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric,fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic,phenylacetic, cinnamic and salicylic acids, as well as sulfonic acidssuch as p-toluene sulfonic and methanesulfonic acids. Either the mono ordi-acid salts can be formed, and such salts may exist in either ahydrated, solvated or substantially anhydrous form. In general, the acidaddition salts of compounds of Formula I are more soluble in water andvarious hydrophilic organic solvents, and generally demonstrate highermelting points in comparison to their free base forms. The selection ofthe appropriate salt will be known to one skilled in the art. Othernon-pharmaceutically acceptable salts, e.g., oxalates, may be used, forexample, in the isolation of compounds of Formula I for laboratory use,or for subsequent conversion to a pharmaceutically acceptable acidaddition salt.

The term “pharmaceutically acceptable basic addition salt” as usedherein means any non-toxic organic or inorganic base addition salt ofany acid compounds represented by Formula I or any of theirintermediates. Illustrative inorganic bases which form suitable saltsinclude lithium, sodium, potassium, calcium, magnesium, or bariumhydroxide. Illustrative organic bases which form suitable salts includealiphatic, alicyclic, or aromatic organic amines such as methylamine,trimethylamine and picoline or ammonia. The selection of the appropriatesalt will be known to a person skilled in the art.

Methods of Treatment

Provided herein are methods of treating elastic fiber related disorder.In certain embodiments, the present disclosure provides methods oftreating elastic fiber related disorder by administering doses oflow-molecular weight hyaluronic acid inhalation solution. Non-limitingexamples of elastic fiber related disorder include cutis laxa, Williamssyndrome, alpha-1 antitrypsin deficiency (elastin is excessivelydegraded by elastase), emphysema, liver disease, Buschke-Ollendorffsyndrome, Menkes disease, pseudoxanthoma elasticum, Marfan's syndromeHurler disease, hypertension and congenital heart defects.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1: Evaluate the Therapeutic Role of HA

To evaluate the therapeutic role of HA, a 2-week, randomized,double-blind, placebo-controlled, phase 2a safety trial was conducted in11 patients with COPD. The primary aims of the study were: 1) todetermine the safety of administering repeated doses of HA to subjectswith smoking-related COPD and 2) to assess the effects of HA on elastindegradation, as measured by sputum and plasma levels of desmosine andisodesmosine (DID), cross-linking amino acids found only in thisprotein. The results indicate that inhalation of HA is well toleratedand rapidly reduces lung elastic fiber breakdown, thus supporting alonger-term investigation of the clinical efficacy of this agent.

Study Protocol

Eleven patients were recruited from 2 centers: 9 from ResearchAssociates in Tucson, Ariz. and 2 from St Luke's-Roosevelt HospitalPulmonary Disease Center in New York. The trial (NCT00993707) wasconducted under IND number 70299, with Institutional Review Boardapproval from both organizations, and complied with the standards ofGood Clinical Practice.

The patients ranged in age from 40 to 76 and fulfilled the diagnosticcriteria of COPD with GOLD grades 2 and 3 with moderate airwayobstruction (forced expiratory volume in 1 second above 40% ofpredicted) and at least a 10 pack-year history of cigarette smoking(Table 1). Ten were white and 1 was African-American. Informed consentwas obtained from all participants.

TABLE 1 Patient characteristics. Characteristic HA Treatment PlaceboNumber of subjects 8 3 Age (years) 63 ± 8^(a)  52 ± 11^(a) Race 7 white,1 African-American 3 white FEV₁ (L) 1.84 ± 0.45^(a) 1.77 ± 0.44^(a) DLCO(mL/min/mmHg) 16.6 ± 8.0^(a)  19.6 ± 0.8^(a)  Total lung capacity (L)7.1 ± 2.1^(a) 5.9 ± 0.5^(a) Smoking Hx >10 pack-years >10 pack-yearsActive smokers None None Inhaled corticosteroids 4 None Recentexacerbations None None Note: ^(a)Mean ± SD. Abbreviations: DLCO, carbonmonoxide diffusing capacity; FEV₁, forced expiratory volume in 1 second;Hx, history; SD, standard deviation.

None were actively smoking at the time of recruitment, and smokingcessation had occurred at least 1 year prior to the study. They wererandomly assigned to treatment groups receiving either 0.01% HA BID (8patients) or matching placebo (3 patients). All patients were instructedto continue their usual bronchodilator therapy during the trial.

Each patient self-administered 3 mL of aerosolized inhalation solution,using a Pari nebulizer, twice daily for 14 days. The primary safety endpoints were oxygen saturation, spirometry, lung volumes, physicalexamination, vital signs, electrocardiogram, and laboratory evaluations(complete blood count, serum chemistries, and urinalysis). Additionally,DID concentrations in sputum and plasma were measured prior to treatmentwith either HA or placebo, and again at weekly intervals, including theweek following treatment.

HA Drug Preparation

The treatment agent used for the study consisted of 0.01% HA isolatedfrom Streptococcus equi (150 kDa weight average molecular weight);dissolved in 3 mL of isotonic, buffered saline; and packaged in plasticvials under sterile conditions. The placebo consisted of identicallypackaged isotonic, buffered saline without HA.

Measurement of DID

Samples were combined with an equal volume of 37% HCl and hydrolyzed at110° C. for 24 h, then dried under vacuum, suspended in 2 mL of butanol,acetic acid, and water (4:1:1), applied to a CF1 column, washed, andeluted. Separation of DID was performed with a 2×100 mm dC18 column(Waters Corporation, Milford, Mass., USA), using a combination of mobilephases: 1) 7 mM heptafluorobutyric acid and 5 mM ammonium acetate inwater and 2) 7 mM heptafluorobutyric acid and 5 mM ammonium acetate in80% acetonitrile. The elution gradient was programmed linearly from 100%A to 90% A over a 10-min interval, and the separated crosslinks wereanalyzed with a TSQ Discovery electrospray tandem mass spectrometer(Thermo Fisher Scientific, Waltham, Mass., USA), using selected reactionmonitoring of mass-to-charge ratio transitions. The results werequantified by comparison with an external d4-labeled desmosine standardand the sputum results were normalized to total protein, as measured bya Micro BCATM assay kit (Thermo Fisher Scientific, Springfield Township,N.J., USA).

Statistical Analysis

Statistically significant correlations (p<0.05) between DID levels andlength of time following treatment were determined by performing atwo-tailed t-test on the Pearson coefficient (r). Results were expressedas mean±standard error of the mean for plasma and sputum DID levels andas mean±standard deviation for all other measurements.

Safety Assessment

The administration of HA had no significant effect on spirometry, lungvolumes, electrocardiograms, and hematological indices. In particular,forced expiratory volume measurements at 1 second showed no significantchanges during the course of the study, which included a 1-week intervalposttreatment (Table 2). Similarly, carbon monoxide diffusing capacityremained unchanged during the 2-week trial (Table 3). Adverse eventswere generally mild and recurred with greater frequency in the placebogroup (Table 4). None could be directly attributed to the inhalationprocedure.

TABLE 2 FEV₁ (liters ± SD). Time point HA^(a) Placebo^(b) p-valuePretreatment 1.84 ± 0.45 1.77 ± 0.44 0.92 Week 1 1.80 ± 0.42 1.75 ± 0.380.92 Week 2 1.72 ± 0.33 1.73 ± 0.31 1.0 Week 3 1.90 ± 0.51 1.70 ± 0.290.36 Note: ^(a)N = 8, ^(b)N = 3. Abbreviations: FEV₁, forced expiratoryvolume in 1 second; SD, standard deviation.

TABLE 3 DLCO (mL/min/mmHg ± SD). Time point HA^(a) Placebo^(b) p-valuePretreatment 16.6 ± 8.0 19.6 ± 0.8 0.54 Week 1 16.2 ± 7.8 19.8 ± 1.50.46 Week 2 15.6 ± 6.9 19.8 ± 1.0 0.34 Note: ^(a)N = 8, ^(b)N = 3.Abbreviations: DLCO, carbon monoxide diffusing capacity; SD, standarddeviation.

TABLE 4 Adverse events. HA Placebo Migraine^(a) Difficulty breathing^(a)Joint pain^(b) Lung pain^(a) Chest congestion^(b) Profuse sweating^(a)Fever^(b) Coughing^(b) Intermittent cough^(b) Light-headedness^(b)Pitting edema^(b) Diarrhea^(b) Light-headedness^(b) Note: ^(a)Moderate,^(b)mild.

Biomarker Measurements

The HA group showed a progressive decrease in plasma DID levels over a3-week period following initiation of treatment (r=−0.98; p=0.02; FIG.1). In contrast, there was no significant reduction in the placebo group(r=−0.70; p=0.30). Sputum DID levels, which specifically reflect lungelastin degradation, also showed a progressive decrease over the sametime interval (r=−0.97; p=0.03; FIG. 2).

Example 2: Evaluate the Therapeutic Role of HA

To further to evaluate the safety and efficacy of administering repeateddoses of Hyaluronic Acid Inhalation Solution to subjects with Emphysemathat have Alpha-1-Antitrypsin deficiency, a randomized, double-blind,placebo-controlled, phase 2 safety trial is conducted. The studyprimarily aims to establish desmosine and isodesmosine concentrations inplasma, sputum and urine measured as markers of elastin degradationsystemically in the lung and also markers of inflammation andfibrinogen.

HA Drug Preparation: Hyaluronic Acid Inhalation Solution

The treatment agent (Hyaluronic acid) used for the study consists of0.03% Hyaluronic Acid dissolved in 3 ml of Inhalation Solution and isadministered twice a day. The placebo consists of the inhalationsolution without HA.

Inclusion Criteria:

Men or women aged 18 through 80 years at the time of consent, Diagnosisof emphysema at screening consistent with National Institutes of Healthguidelines 19 GOLD COPD classification stages I, II or III, Evidence ofemphysema on radiographic imaging, A ratio of pre-bronchodilator FEV toforced vital capacity (FVC) of ≤80% at screening, FEV1 ≥30% and ≤79%(post-bronchodilator) of predicted normal at screening, Clinicallaboratory tests (complete blood count, serum chemistry, and urinalysis)within normal limits or clinically acceptable to the PI and sponsor atscreening, Evidence of alpha-1 antitrypsin deficiency (AATD) with anygenotype except PiMZ deficiency, Patients must have stopped usingIntravenous alpha-1 antitrypsin protein (AAT) augmentation therapy atleast 3 months before entering study.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EOUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

We claim: 1-17. (canceled)
 18. A pharmaceutical composition comprising:hyaluronic acid, or a pharmaceutically acceptable salt thereof, whereinthe hyaluronic acid has a weight average molecular weight (M_(w)) ofabout 50 to about 1000 kDa; and a pharmaceutically acceptable excipient,wherein the hyaluronic acid is formulated as a solution of 0.01% to0.06% w/v.
 19. The pharmaceutical composition of claim 1, wherein thehyaluronic acid is formulated as a solution of 0.01% to 0.03% w/v. 20.The pharmaceutical composition of claim 1, wherein the hyaluronic acidis formulated as a solution of 0.01% w/v.
 21. The pharmaceuticalcomposition of claim 1, wherein the hyaluronic acid has a weight averagemolecular weight (M_(w)) of about 150 kDa.
 22. The pharmaceuticalcomposition of claim 1, wherein the hyaluronic acid is formulated forinhalation.
 23. The pharmaceutical composition of claim 22, wherein thehyaluronic acid is formulated for nasal, intrapulmonary, intratracheal,intrabronchial, or intra-alveolar administration.
 24. The pharmaceuticalcomposition of claim 23, wherein the hyaluronic acid is formulated forintra-alveolar administration.
 25. The pharmaceutical composition ofclaim 1, wherein the hyaluronic acid is formulated for administrationthrough a liquid spray device.
 26. The pharmaceutical composition ofclaim 25, wherein the liquid spray device is an aerosol device.
 27. Thepharmaceutical composition of claim 26, wherein the aerosol device is anebulizer or electrohydrodynamic aerosol device.
 28. An inhalercomprising the pharmaceutical composition of claim
 1. 29. The inhaler ofclaim 28, wherein the inhaler is configured to deliver droplets with asize of about 2 μm to about 3 μm.
 30. The inhaler of claim 28, whereinthe inhaler comprises 3 ml of the solution of 0.01% to 0.06% w/v ofhyaluronic acid.
 31. The inhaler of claim 28, wherein the hyaluronicacid is formulated as a solution of 0.01% to 0.03% w/v.
 32. The inhalerof claim 28, wherein the hyaluronic acid is formulated as a solution of0.01% w/v.
 33. The inhaler of claim 28, wherein the inhaler is a liquidspray device.
 34. The inhaler of claim 33, wherein the liquid spraydevice is an aerosol device.
 35. The inhaler of claim 34, wherein theaerosol device is a nebulizer or electrohydrodynamic aerosol device.